Proceedings of the National Academy of Sciences, 1987
Two monoclonal antibodies (OKT27 and OKT27b) have been produced that react with distinct epitopes... more Two monoclonal antibodies (OKT27 and OKT27b) have been produced that react with distinct epitopes of a 95-kDa peptide. The T27 antigen is widely distributed, being expressed on B lymphocytes, monocytes, and adult T-
TCR gene therapy is adversely affected by newly formed TCR␣ heterodimers comprising exogenous an... more TCR gene therapy is adversely affected by newly formed TCR␣ heterodimers comprising exogenous and endogenous TCR chains that dilute expression of transgenic TCR␣ dimers and are potentially self-reactive. We have addressed TCR mispairing by using a modified two-chain TCR that encompasses total human CD3 with specificities for three different Ags. Transfer of either TCR␣:CD3 or :CD3 genes alone does not result in surface expression, whereas transfer of both modified TCR chains results in high surface expression, binding of peptide-MHC complexes and Ag-specific T cell functions. Genetic introduction of TCR␣: does not compromise surface expression and functions of an endogenous TCR␣. Flow cytometry fluorescence resonance energy transfer and biochemical analyses demonstrate that TCR␣:CD3 is the first strategy that results in highly preferred pairing between CD3-modified TCR␣ and  chains as well as absence of TCR mispairing between TCR:CD3 and nonmodified TCR chains. Intracellular assembly and surface expression of TCR:CD3 chains is independent of endogenous CD3␥, ␦, and . Taken together, our data support the use of TCR␣:CD3 to prevent TCR mispairing, which may provide an adequate strategy to enhance efficacy and safety of TCR gene transfer.
The chromatin structure of polyamine-depleted U-87 MG human brain tumour cells was studied by fol... more The chromatin structure of polyamine-depleted U-87 MG human brain tumour cells was studied by following the kinetics of digestion of cell nuclei by micrococcal nuclease and bovine pancreatic DNAase I. Cells growing in monolayers were treated with either alpha-difluoromethylornithine (DFMO), to deplete putrescine and spermidine, or N1,N14-bis(ethyl)homospermine (BE-4-4-4), to deplete putrescine, spermidine and spermine. BE-4-4-4 increased the initial rates of digestion and the magnitudes of limit digest by both enzymes; DFMO increased the limit digests without affecting initial digestion rates. Addition of 1 mM-putrescine 1 day after addition of DFMO reversed the effect of DFMO on limit digests. (Because polyamine uptake is low in cells treated with BE-4-4-4, and because putrescine does not reverse the growth-inhibitory effects of BE-4-4-4, reversal of the effects of BE-4-4-4 with putrescine was not attempted.) The increases in initial rates and limit digests did not result from chan...
The interactions of spermine and polyamine analogues with synthetic polynucleotides of various ba... more The interactions of spermine and polyamine analogues with synthetic polynucleotides of various base sequences complexed with ethidium bromide (EB) were investigated using measurements of fluorescence intensity and steady-state fluorescence polarization. Spermine and polyamine analogues displaced some but not all of the EB bound to poly(dA-dT).poly(dA-dT) or poly(dG-dC).poly(dG-dC), suggesting that polyamines may stabilize these polynucleotides in a conformation with reduced affinity for EB. Modifications of the aliphatic backbone of spermine have pronounced effects on its ability to displace EB from poly(dA-dT).poly(dA-dT) but not from poly-(dG-dC).poly(dG-dC). Spermine and some but not all of the polyamine analogues caused fluorescence depolarization when they interacted with the complex of EB and poly(dA-dT).poly-(dA-dT). Neither spermine nor any of the analogues, however, induced fluorescence depolarization in the complex of EB with poly(dG-dC).poly(dG-dC) or poly(dA).poly(dT). T...
Besides flow cytometry, fluorescence microscopy combined with computerized image analysis offers ... more Besides flow cytometry, fluorescence microscopy combined with computerized image analysis offers an alternative tool for assessing phagocyte oxidant generation at the single-cell level. This technique provides an opportunity for the direct visualization of cells and simultaneous measurement of cellular fluorescence intensity. Thus, we developed a simple method for the quantitative evaluation of intracellular superoxide anion and hydrogen peroxide production with image cytometry by using hydroethidine and dihydrorhodamine 123 dyes, respectively. Human neutrophils stimulated with phorbol dibutyrate and labeled by these fluorogenic substrates showed intense, well recognizable red or green fluorescence. The intensity of signals from individual granulocytes of cytospin preparations were quantitatively measured in digitized images. There was a great heterogeneity in response to the stimulus within the granulocyte population as shown by the integrated fluorescence intensity values. In agreement with the results of parallel flow cytometric experiments, this simple image analysis performed on cells of cytospin preparations was able to detect the defects in the oxidative metabolism of neutrophils from pa-tients with cervix carcinoma. We demonstrated that even minor alterations in superoxide anion/hydrogen peroxide generation can be detected by image cytometry as efficiently as by flow cytometry. This result validates imaging microscopy as an alternative to flow cytometry in such experiments. In addition, the image cytometric technique allows the observation of the kinetics of free radical production in individual cell under adherent conditions. Therefore, we carried out image analysis of the oxidative burst of neutrophils adherent to uncoated glass and fibronectin-and type IV collagen-coated surfaces in response to stimulation with phorbol dibutyrate or N-formyl-methionyl-leucyl-phenylalanine. We elaborated a calibration technique for the quantitative measurement of the ethidium bromide generation mediated by superoxide anion within individual adherent granulocytes. The ethidium bromide production varied between 0.48 and 1.17 amol/cell/min. Cytometry 33:19-31, 1998.
Trastuzumab resistance CD44 Hyaluronan Masking A B S T R A C T Although trastuzumab, a recombinan... more Trastuzumab resistance CD44 Hyaluronan Masking A B S T R A C T Although trastuzumab, a recombinant humanised anti-ErbB2 antibody, is widely used in the treatment of breast cancer, neither its mechanism of action, nor the factors leading to resistance are fully understood. We have previously shown that antibody-dependent cellular cytotoxicity is pivotal in the in vivo effect of trastuzumab against JIMT-1, a cell line showing in vitro resistance to the antibody, and suggested that masking of the trastuzumab-binding epitope by MUC-4, a cell surface mucin, took place. Here, we further explored the role of masking of ErbB2 in connection with CD44 expression and synthesis of its ligand, hyaluronan. We show that high expression of CD44 observed in JIMT-1 cells correlates with ErbB2 downregulation in vivo, while siRNA-mediated inhibition of CD44 expression leads to decreased rate of trastuzumab internalisation and low cell proliferation in vitro. An inhibitor of hyaluronan synthesis, 4-methylumbelliferon (4-MU) significantly reduced the hyaluronan level of JIMT-1 cells both in vivo and in vitro leading to enhanced binding of trastuzumab to ErbB2 and increased ErbB2 down-regulation. Furthermore, the inhibitory effect of trastuzumab on the growth of JIMT-1 xenografts was significantly increased by 4-MU treatment. Our results point to the importance of the CD44-hyaluronan pathway in the escape of tumour cells from receptor-oriented therapy.
A concise review is presented on the nature, possible origin and functional significance of cell ... more A concise review is presented on the nature, possible origin and functional significance of cell surface receptor patterns in the plasma membrane of lymphoid cells. A special emphasize has been laid on the available methodological approaches, their individual virtues and sources of errors. Fluorescence energy transfer is one of the oldest available means for studying non-randomized co-distribution patterns of cell surface receptors. A detailed and critical description is given on the generation of two-dimensional cell surface receptor patterns based on pair-wise energy transfer measurements. A second hierarchical-level of receptor clusters have been described by electron and scanning force microscopies after immuno-gold-labeling of distinct receptor kinds. The origin of these receptor Ž . islands at a nanometer scale and island groups at a higher hierarchical m level, has been explained mostly by Ž . detergent insoluble glycolipid-enriched complexes known as rafts, or detergent insoluble glycolipids DIGs . These w rafts are the most-likely organizational forces behind at least some kind of receptor clustering K. Simons et al., Ž . x Nature 387 1997 569 . These models, which have great significance in trans-membrane signaling and intra-membrane and intracellular trafficking, are accentuating the necessity to revisit the Singer᎐Nicolson fluid mosaic w membrane model and substitute the free protein diffusion with a restricted diffusion concept S.J. Singer et al., Ž . x Science 175 1972 720 .
The partition coefficient, surface activity and membrane fluidizing/disordering effects of CH-103... more The partition coefficient, surface activity and membrane fluidizing/disordering effects of CH-103, a beta-adrenergic receptor antagonist, were compared to those of propranolol and practolol as reference compounds. Changes in membrane fluidity were followed by measuring the steady-state fluorescence anisotropy of bull sperm cells with 1-[4-(trimethylammonium)-phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) as a fluorescence probe. The octanol/buffer (pH 7.0) partition coefficients for CH-103, propranolol and practolol were 32.9, 5.08 and 0.013, respectively; the surface activity of the compounds decreased in the same order. CH-103 and propranolol significantly increased the fluidity of the membrane in a concentration-dependent manner, whereas practolol reduced fluidity. These physicochemical parameters correlated with the effects of these drugs on rat sarcolemmal Ca2+, Mg(2+)-ATPase, a manifestation of their nonspecific membrane activity. Our results suggest that the physicochemical properties of CH-103, similarly to those of propranolol, are the main determinants of its nonspecific membrane activity.
Lentinan, an immunopotentiating polysaccharide, stimulated the pinocytosis of horseradish peroxid... more Lentinan, an immunopotentiating polysaccharide, stimulated the pinocytosis of horseradish peroxidase (HRP) or FITC-dextran by resident or thioglycollate-elicited mouse macrophages from 10 to 50% in a dose dependent manner. Pinocytosis of HRP and FITC-dextran by C4M phi cells, a murine macrophage cell line, exhibiting a lower basic pinocytic activity than peritoneal cells, was augmented up to 310 and 120%, respectively, by lentinan. Mannan inhibited the HRP uptake by peritoneal macrophages via specific mannose receptors. This inhibitory effect was partly abolished, when lentinan was also added to the cells. Mannan was not able to inhibit pinocytosis of HRP by C4M phi macrophages, indicating little or no mannose receptor activity on these cells. Pinocytosis of FITC-dextran was not affected by mannan. Lentinan, opsonized in mouse sera inhibited the uptake of HRP by peritoneal macrophages by 30-35%. Opsonized lentinan and mannan added together caused 60% inhibition of HRP uptake in peri...
Lentinan, an immunopotentiating beta-1,3-glucan polysaccharide stimulated the in vitro phagocytos... more Lentinan, an immunopotentiating beta-1,3-glucan polysaccharide stimulated the in vitro phagocytosis of BSA-coated, C3b- or monoclonal immunoglobulin (IgG2b)-coated fluorescent microspheres by resident or thioglycollate-elicited mouse macrophages in a dose-dependent manner. Analysis of flow cytometric data has shown that microbead phagocytosis of resident macrophages, which exhibit a lower basic phagocytic activity than the thioglycollate elicited ones, has been augmented by up to 900% due to lentinan. The percent ratio of phagocytes among peritoneal exudate cells, however, remained unchanged after short-term lentinan stimulation. Preincubation of the cells with lentinan resulted in increased ingestion of the microbeads. Activation of phagocytosis by lentinan is therefore due in part to the direct stimulation of the cells, however, lentinan also serves as supplementary opsonin for C3b-coated beads. Mannan inhibited the ingestion of C3b-coated microspheres by 75%, which was abolished ...
The phagocytosis of uniform fluorescent latex particles by resident and thioglycollate-elicited m... more The phagocytosis of uniform fluorescent latex particles by resident and thioglycollate-elicited macrophages was analysed by flow cytometry. The percentage of phagocytosing macrophages and the number of internalized microspheres per cell was determined from cell size and fluorescence histograms. Results were corrected for the adherence of microbeads to the cells in the presence of sodium azide in the medium. Human C3b- or murine monoclonal IgG-coated microspheres were applied to assess receptor-mediated phagocytosis in different inbred strains of mice. Phagocytic activity of thioglycollate-elicited macrophages was consequently higher than that of resident macrophages. A decreasing gradient of C3b and Fc receptor-mediated phagocytosis was established in the following order: B10.BR, B10, C3H/Di and C3H.SW strains. Our results indicate that the phagocytic function of murine macrophages is under control of both the somatic (non-H-2) and H-2 genes.
The costs of publication of this article were defrayed in pari by the payment of accordance wlth ... more The costs of publication of this article were defrayed in pari by the payment of accordance wlth 18 U.S.C. Section 1734 solely to indicate this fact.
Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resu... more Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1balpha (GP1balpha). PDI contains two active site dithiols/disulfides. The active sites of 26% of the PDI on resting platelets was in the dithiol form, compared with 81% in the dithiol form on activated platelets. Similarly, GP1balpha presented one or more free thiols on the activated platelet surface but not on resting platelets. Anti-PDI antibodies increased the dissociation constant for binding of vWF to platelets by approximately 50% and PDI and GP1balpha were sufficiently close on the platelet surface to allow fluorescence resonance energy transfer between chromophores attached to PDI and GP1balpha. Incubation of resting platelets with anti-PDI antibodies followed by activation with thrombin enhanced labeling and binding of monoclonal antibodies to the N-terminal region of GP1balpha on the activated platelet surface. These observations indicated that platelet activation triggered reduction of the active site disulfides of PDI and a conformational change in GP1balpha that resulted in exposure of a free thiol(s).
a-Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme ... more a-Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, inhibits the growth of brain tumor cell lines and is undergoing clinical trials as a treatment for brain tumors. Platelet-derived growth factor (PDGF) is thought to regulate the growth and development of precursors of both normal and neoplastic astrocytic cells; calcium signaling is thought to play a role in the transduction of PDGF signals. Using laser fluorescence image cytometry, flow cytometry, and spectrofluorometry, we studied the effect of DFMO on the calcium signals induced by PDGF in A172 human glioblastoma cells. Four days of treatment with 5 mM DFMO substantially shortened PDGF-induced calcium signals. The effect was reversed more than 10 h hut less than 24 h after putrescine treatment, even though polyamines were repleted 4 h after putrescine and spermidine were added. DFMO did not substantially affect intracellular calcium release or the timing of the opening and closing of plasma membrane calcium channels. These findings support the notion that calcium signaling may be a target for inhibitors of polyamine metabolism.
ErbB2 (HER2, Neu), a member of the epidermal growth factor (EGF) receptor tyrosine kinase family,... more ErbB2 (HER2, Neu), a member of the epidermal growth factor (EGF) receptor tyrosine kinase family, is often overexpressed in breast cancer and other malignancies. ErbB2 homodimerizes but also presents as a common auxiliary subunit of the EGF and heregulin receptors (erbB1 or EGFR; and erbB3-4, respectively), with which it heteroassociates. ErbB2 is generally regarded as an orphan (ligand-less) receptor with a very potent kinase domain activated either via its associated partners or constitutively as a consequence of discrete mutations. It follows that the extent and regulation of its cell surface interactions are of central importance. We have studied the large-scale association pattern of erbB2 in quiescent and activated cells labeled with fluorescent anti-erbB2 monoclonal antibodies using scanning near-field optical microscopy (SNOM). ErbB2 was found to be concentrated in irregular membrane patches with a mean diameter of approx. 0.5 microm in nonactivated SKBR3 and MDA453 human br...
We have investigated the molecular basis for these effects by comparing the protein tyrosine phos... more We have investigated the molecular basis for these effects by comparing the protein tyrosine phosphorylation signals induced by Campath-1H and the CD3 mAb OKT3 in primary T cells, and in CD45 ⍣ TCR ⍣ , CD45 -TCR ⍣ and CD45 ⍣ TCR -Jurkat subclones transfected with CD52. Our results show that Campath-1H triggers similar tyrosine phosphorylation events as OKT3 in both primary T cells and in the CD45 ⍣ TCR ⍣ Jurkat sub-clone, albeit at quantitatively lower levels. However, no phospholipase Cγ1 activation nor calcium signals were detected in response to CD52 ligation. The CD52-mediated induction of protein tyrosine phosphorylation was absolutely dependent upon the expression of both the TCR and the CD45 phosphotyrosine phosphatase at the cell surface. Cross-linking of Campath-1H was essential for signal transduction in all cells investigated. Fluorescence resonance energy transfer was used to demonstrate CD52 homo-association at the cell surface in Jurkat T cells in a TCR-and CD45-independent manner, and CD52-TCR association in CD45 ⍣ TCR ⍣ cells. We propose a model to explain the activating effects of Campath-1H in which CD52 mAb cross-linking causes the trapping of TCR polypeptides within molecular complexes at the cell surface, thereby inducing signals via the TCR by a process which depends on the CD45-mediated regulation of the p56 lck and p59 fyn tyrosine kinases.
An investigation into the role of CD45 isoforms in T cell antigen receptor signal transduction wa... more An investigation into the role of CD45 isoforms in T cell antigen receptor signal transduction was carried out by transfecting CD45-negative CD4 ؉ CD8 ؉ HPB-ALL T cells with the CD45R0, CD45RBC, and CD45RABC isoforms. Fluorescence resonance energy transfer analysis showed that the CD45R0 isoform, but not the CD45RBC or CD45RABC isoforms, was found as homodimers and also preferentially associated with CD4 and CD8 at the cell-surface. A comparison was therefore made of T cell antigen receptor signaling between sub-clones expressing either CD45R0 or CD45RBC. Under basal conditions CD4-associated p56 lck tyrosine kinase activity and cellular protein tyrosine phosphorylation levels were higher in the CD45R0 ؉ than in the CD45RBC ؉ sub-clones. Upon CD3-CD4 ligation, TCR-phosphorylation, ZAP-70 recruitment to the p21/p23 TCR-phosphoisomers, ZAP-70 phosphorylation, as well as p56 lck , c-Cbl and Slp-76 phosphorylation, were all markedly increased in CD45R0 ؉ compared with CD45RBC ؉ cells. T cell antigen receptor (TCR) stimulation alone also promoted c-Cbl phosphorylation in CD45R0 ؉ but not in CD45RBC ؉ cells. Our results are consistent with a model in which association of CD45R0 with CD4 generates a more active pool of CD4-associated p56 lck kinase molecules. Upon CD3-CD4 co-ligation, the active p56 lck increases the intensity of T cell antigen receptor signal transduction coupling by promoting TCR-chain phosphorylation and ZAP-70 recruitment.
Proceedings of the National Academy of Sciences, 1987
Two monoclonal antibodies (OKT27 and OKT27b) have been produced that react with distinct epitopes... more Two monoclonal antibodies (OKT27 and OKT27b) have been produced that react with distinct epitopes of a 95-kDa peptide. The T27 antigen is widely distributed, being expressed on B lymphocytes, monocytes, and adult T-
TCR gene therapy is adversely affected by newly formed TCR␣ heterodimers comprising exogenous an... more TCR gene therapy is adversely affected by newly formed TCR␣ heterodimers comprising exogenous and endogenous TCR chains that dilute expression of transgenic TCR␣ dimers and are potentially self-reactive. We have addressed TCR mispairing by using a modified two-chain TCR that encompasses total human CD3 with specificities for three different Ags. Transfer of either TCR␣:CD3 or :CD3 genes alone does not result in surface expression, whereas transfer of both modified TCR chains results in high surface expression, binding of peptide-MHC complexes and Ag-specific T cell functions. Genetic introduction of TCR␣: does not compromise surface expression and functions of an endogenous TCR␣. Flow cytometry fluorescence resonance energy transfer and biochemical analyses demonstrate that TCR␣:CD3 is the first strategy that results in highly preferred pairing between CD3-modified TCR␣ and  chains as well as absence of TCR mispairing between TCR:CD3 and nonmodified TCR chains. Intracellular assembly and surface expression of TCR:CD3 chains is independent of endogenous CD3␥, ␦, and . Taken together, our data support the use of TCR␣:CD3 to prevent TCR mispairing, which may provide an adequate strategy to enhance efficacy and safety of TCR gene transfer.
The chromatin structure of polyamine-depleted U-87 MG human brain tumour cells was studied by fol... more The chromatin structure of polyamine-depleted U-87 MG human brain tumour cells was studied by following the kinetics of digestion of cell nuclei by micrococcal nuclease and bovine pancreatic DNAase I. Cells growing in monolayers were treated with either alpha-difluoromethylornithine (DFMO), to deplete putrescine and spermidine, or N1,N14-bis(ethyl)homospermine (BE-4-4-4), to deplete putrescine, spermidine and spermine. BE-4-4-4 increased the initial rates of digestion and the magnitudes of limit digest by both enzymes; DFMO increased the limit digests without affecting initial digestion rates. Addition of 1 mM-putrescine 1 day after addition of DFMO reversed the effect of DFMO on limit digests. (Because polyamine uptake is low in cells treated with BE-4-4-4, and because putrescine does not reverse the growth-inhibitory effects of BE-4-4-4, reversal of the effects of BE-4-4-4 with putrescine was not attempted.) The increases in initial rates and limit digests did not result from chan...
The interactions of spermine and polyamine analogues with synthetic polynucleotides of various ba... more The interactions of spermine and polyamine analogues with synthetic polynucleotides of various base sequences complexed with ethidium bromide (EB) were investigated using measurements of fluorescence intensity and steady-state fluorescence polarization. Spermine and polyamine analogues displaced some but not all of the EB bound to poly(dA-dT).poly(dA-dT) or poly(dG-dC).poly(dG-dC), suggesting that polyamines may stabilize these polynucleotides in a conformation with reduced affinity for EB. Modifications of the aliphatic backbone of spermine have pronounced effects on its ability to displace EB from poly(dA-dT).poly(dA-dT) but not from poly-(dG-dC).poly(dG-dC). Spermine and some but not all of the polyamine analogues caused fluorescence depolarization when they interacted with the complex of EB and poly(dA-dT).poly-(dA-dT). Neither spermine nor any of the analogues, however, induced fluorescence depolarization in the complex of EB with poly(dG-dC).poly(dG-dC) or poly(dA).poly(dT). T...
Besides flow cytometry, fluorescence microscopy combined with computerized image analysis offers ... more Besides flow cytometry, fluorescence microscopy combined with computerized image analysis offers an alternative tool for assessing phagocyte oxidant generation at the single-cell level. This technique provides an opportunity for the direct visualization of cells and simultaneous measurement of cellular fluorescence intensity. Thus, we developed a simple method for the quantitative evaluation of intracellular superoxide anion and hydrogen peroxide production with image cytometry by using hydroethidine and dihydrorhodamine 123 dyes, respectively. Human neutrophils stimulated with phorbol dibutyrate and labeled by these fluorogenic substrates showed intense, well recognizable red or green fluorescence. The intensity of signals from individual granulocytes of cytospin preparations were quantitatively measured in digitized images. There was a great heterogeneity in response to the stimulus within the granulocyte population as shown by the integrated fluorescence intensity values. In agreement with the results of parallel flow cytometric experiments, this simple image analysis performed on cells of cytospin preparations was able to detect the defects in the oxidative metabolism of neutrophils from pa-tients with cervix carcinoma. We demonstrated that even minor alterations in superoxide anion/hydrogen peroxide generation can be detected by image cytometry as efficiently as by flow cytometry. This result validates imaging microscopy as an alternative to flow cytometry in such experiments. In addition, the image cytometric technique allows the observation of the kinetics of free radical production in individual cell under adherent conditions. Therefore, we carried out image analysis of the oxidative burst of neutrophils adherent to uncoated glass and fibronectin-and type IV collagen-coated surfaces in response to stimulation with phorbol dibutyrate or N-formyl-methionyl-leucyl-phenylalanine. We elaborated a calibration technique for the quantitative measurement of the ethidium bromide generation mediated by superoxide anion within individual adherent granulocytes. The ethidium bromide production varied between 0.48 and 1.17 amol/cell/min. Cytometry 33:19-31, 1998.
Trastuzumab resistance CD44 Hyaluronan Masking A B S T R A C T Although trastuzumab, a recombinan... more Trastuzumab resistance CD44 Hyaluronan Masking A B S T R A C T Although trastuzumab, a recombinant humanised anti-ErbB2 antibody, is widely used in the treatment of breast cancer, neither its mechanism of action, nor the factors leading to resistance are fully understood. We have previously shown that antibody-dependent cellular cytotoxicity is pivotal in the in vivo effect of trastuzumab against JIMT-1, a cell line showing in vitro resistance to the antibody, and suggested that masking of the trastuzumab-binding epitope by MUC-4, a cell surface mucin, took place. Here, we further explored the role of masking of ErbB2 in connection with CD44 expression and synthesis of its ligand, hyaluronan. We show that high expression of CD44 observed in JIMT-1 cells correlates with ErbB2 downregulation in vivo, while siRNA-mediated inhibition of CD44 expression leads to decreased rate of trastuzumab internalisation and low cell proliferation in vitro. An inhibitor of hyaluronan synthesis, 4-methylumbelliferon (4-MU) significantly reduced the hyaluronan level of JIMT-1 cells both in vivo and in vitro leading to enhanced binding of trastuzumab to ErbB2 and increased ErbB2 down-regulation. Furthermore, the inhibitory effect of trastuzumab on the growth of JIMT-1 xenografts was significantly increased by 4-MU treatment. Our results point to the importance of the CD44-hyaluronan pathway in the escape of tumour cells from receptor-oriented therapy.
A concise review is presented on the nature, possible origin and functional significance of cell ... more A concise review is presented on the nature, possible origin and functional significance of cell surface receptor patterns in the plasma membrane of lymphoid cells. A special emphasize has been laid on the available methodological approaches, their individual virtues and sources of errors. Fluorescence energy transfer is one of the oldest available means for studying non-randomized co-distribution patterns of cell surface receptors. A detailed and critical description is given on the generation of two-dimensional cell surface receptor patterns based on pair-wise energy transfer measurements. A second hierarchical-level of receptor clusters have been described by electron and scanning force microscopies after immuno-gold-labeling of distinct receptor kinds. The origin of these receptor Ž . islands at a nanometer scale and island groups at a higher hierarchical m level, has been explained mostly by Ž . detergent insoluble glycolipid-enriched complexes known as rafts, or detergent insoluble glycolipids DIGs . These w rafts are the most-likely organizational forces behind at least some kind of receptor clustering K. Simons et al., Ž . x Nature 387 1997 569 . These models, which have great significance in trans-membrane signaling and intra-membrane and intracellular trafficking, are accentuating the necessity to revisit the Singer᎐Nicolson fluid mosaic w membrane model and substitute the free protein diffusion with a restricted diffusion concept S.J. Singer et al., Ž . x Science 175 1972 720 .
The partition coefficient, surface activity and membrane fluidizing/disordering effects of CH-103... more The partition coefficient, surface activity and membrane fluidizing/disordering effects of CH-103, a beta-adrenergic receptor antagonist, were compared to those of propranolol and practolol as reference compounds. Changes in membrane fluidity were followed by measuring the steady-state fluorescence anisotropy of bull sperm cells with 1-[4-(trimethylammonium)-phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) as a fluorescence probe. The octanol/buffer (pH 7.0) partition coefficients for CH-103, propranolol and practolol were 32.9, 5.08 and 0.013, respectively; the surface activity of the compounds decreased in the same order. CH-103 and propranolol significantly increased the fluidity of the membrane in a concentration-dependent manner, whereas practolol reduced fluidity. These physicochemical parameters correlated with the effects of these drugs on rat sarcolemmal Ca2+, Mg(2+)-ATPase, a manifestation of their nonspecific membrane activity. Our results suggest that the physicochemical properties of CH-103, similarly to those of propranolol, are the main determinants of its nonspecific membrane activity.
Lentinan, an immunopotentiating polysaccharide, stimulated the pinocytosis of horseradish peroxid... more Lentinan, an immunopotentiating polysaccharide, stimulated the pinocytosis of horseradish peroxidase (HRP) or FITC-dextran by resident or thioglycollate-elicited mouse macrophages from 10 to 50% in a dose dependent manner. Pinocytosis of HRP and FITC-dextran by C4M phi cells, a murine macrophage cell line, exhibiting a lower basic pinocytic activity than peritoneal cells, was augmented up to 310 and 120%, respectively, by lentinan. Mannan inhibited the HRP uptake by peritoneal macrophages via specific mannose receptors. This inhibitory effect was partly abolished, when lentinan was also added to the cells. Mannan was not able to inhibit pinocytosis of HRP by C4M phi macrophages, indicating little or no mannose receptor activity on these cells. Pinocytosis of FITC-dextran was not affected by mannan. Lentinan, opsonized in mouse sera inhibited the uptake of HRP by peritoneal macrophages by 30-35%. Opsonized lentinan and mannan added together caused 60% inhibition of HRP uptake in peri...
Lentinan, an immunopotentiating beta-1,3-glucan polysaccharide stimulated the in vitro phagocytos... more Lentinan, an immunopotentiating beta-1,3-glucan polysaccharide stimulated the in vitro phagocytosis of BSA-coated, C3b- or monoclonal immunoglobulin (IgG2b)-coated fluorescent microspheres by resident or thioglycollate-elicited mouse macrophages in a dose-dependent manner. Analysis of flow cytometric data has shown that microbead phagocytosis of resident macrophages, which exhibit a lower basic phagocytic activity than the thioglycollate elicited ones, has been augmented by up to 900% due to lentinan. The percent ratio of phagocytes among peritoneal exudate cells, however, remained unchanged after short-term lentinan stimulation. Preincubation of the cells with lentinan resulted in increased ingestion of the microbeads. Activation of phagocytosis by lentinan is therefore due in part to the direct stimulation of the cells, however, lentinan also serves as supplementary opsonin for C3b-coated beads. Mannan inhibited the ingestion of C3b-coated microspheres by 75%, which was abolished ...
The phagocytosis of uniform fluorescent latex particles by resident and thioglycollate-elicited m... more The phagocytosis of uniform fluorescent latex particles by resident and thioglycollate-elicited macrophages was analysed by flow cytometry. The percentage of phagocytosing macrophages and the number of internalized microspheres per cell was determined from cell size and fluorescence histograms. Results were corrected for the adherence of microbeads to the cells in the presence of sodium azide in the medium. Human C3b- or murine monoclonal IgG-coated microspheres were applied to assess receptor-mediated phagocytosis in different inbred strains of mice. Phagocytic activity of thioglycollate-elicited macrophages was consequently higher than that of resident macrophages. A decreasing gradient of C3b and Fc receptor-mediated phagocytosis was established in the following order: B10.BR, B10, C3H/Di and C3H.SW strains. Our results indicate that the phagocytic function of murine macrophages is under control of both the somatic (non-H-2) and H-2 genes.
The costs of publication of this article were defrayed in pari by the payment of accordance wlth ... more The costs of publication of this article were defrayed in pari by the payment of accordance wlth 18 U.S.C. Section 1734 solely to indicate this fact.
Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resu... more Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1balpha (GP1balpha). PDI contains two active site dithiols/disulfides. The active sites of 26% of the PDI on resting platelets was in the dithiol form, compared with 81% in the dithiol form on activated platelets. Similarly, GP1balpha presented one or more free thiols on the activated platelet surface but not on resting platelets. Anti-PDI antibodies increased the dissociation constant for binding of vWF to platelets by approximately 50% and PDI and GP1balpha were sufficiently close on the platelet surface to allow fluorescence resonance energy transfer between chromophores attached to PDI and GP1balpha. Incubation of resting platelets with anti-PDI antibodies followed by activation with thrombin enhanced labeling and binding of monoclonal antibodies to the N-terminal region of GP1balpha on the activated platelet surface. These observations indicated that platelet activation triggered reduction of the active site disulfides of PDI and a conformational change in GP1balpha that resulted in exposure of a free thiol(s).
a-Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme ... more a-Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, inhibits the growth of brain tumor cell lines and is undergoing clinical trials as a treatment for brain tumors. Platelet-derived growth factor (PDGF) is thought to regulate the growth and development of precursors of both normal and neoplastic astrocytic cells; calcium signaling is thought to play a role in the transduction of PDGF signals. Using laser fluorescence image cytometry, flow cytometry, and spectrofluorometry, we studied the effect of DFMO on the calcium signals induced by PDGF in A172 human glioblastoma cells. Four days of treatment with 5 mM DFMO substantially shortened PDGF-induced calcium signals. The effect was reversed more than 10 h hut less than 24 h after putrescine treatment, even though polyamines were repleted 4 h after putrescine and spermidine were added. DFMO did not substantially affect intracellular calcium release or the timing of the opening and closing of plasma membrane calcium channels. These findings support the notion that calcium signaling may be a target for inhibitors of polyamine metabolism.
ErbB2 (HER2, Neu), a member of the epidermal growth factor (EGF) receptor tyrosine kinase family,... more ErbB2 (HER2, Neu), a member of the epidermal growth factor (EGF) receptor tyrosine kinase family, is often overexpressed in breast cancer and other malignancies. ErbB2 homodimerizes but also presents as a common auxiliary subunit of the EGF and heregulin receptors (erbB1 or EGFR; and erbB3-4, respectively), with which it heteroassociates. ErbB2 is generally regarded as an orphan (ligand-less) receptor with a very potent kinase domain activated either via its associated partners or constitutively as a consequence of discrete mutations. It follows that the extent and regulation of its cell surface interactions are of central importance. We have studied the large-scale association pattern of erbB2 in quiescent and activated cells labeled with fluorescent anti-erbB2 monoclonal antibodies using scanning near-field optical microscopy (SNOM). ErbB2 was found to be concentrated in irregular membrane patches with a mean diameter of approx. 0.5 microm in nonactivated SKBR3 and MDA453 human br...
We have investigated the molecular basis for these effects by comparing the protein tyrosine phos... more We have investigated the molecular basis for these effects by comparing the protein tyrosine phosphorylation signals induced by Campath-1H and the CD3 mAb OKT3 in primary T cells, and in CD45 ⍣ TCR ⍣ , CD45 -TCR ⍣ and CD45 ⍣ TCR -Jurkat subclones transfected with CD52. Our results show that Campath-1H triggers similar tyrosine phosphorylation events as OKT3 in both primary T cells and in the CD45 ⍣ TCR ⍣ Jurkat sub-clone, albeit at quantitatively lower levels. However, no phospholipase Cγ1 activation nor calcium signals were detected in response to CD52 ligation. The CD52-mediated induction of protein tyrosine phosphorylation was absolutely dependent upon the expression of both the TCR and the CD45 phosphotyrosine phosphatase at the cell surface. Cross-linking of Campath-1H was essential for signal transduction in all cells investigated. Fluorescence resonance energy transfer was used to demonstrate CD52 homo-association at the cell surface in Jurkat T cells in a TCR-and CD45-independent manner, and CD52-TCR association in CD45 ⍣ TCR ⍣ cells. We propose a model to explain the activating effects of Campath-1H in which CD52 mAb cross-linking causes the trapping of TCR polypeptides within molecular complexes at the cell surface, thereby inducing signals via the TCR by a process which depends on the CD45-mediated regulation of the p56 lck and p59 fyn tyrosine kinases.
An investigation into the role of CD45 isoforms in T cell antigen receptor signal transduction wa... more An investigation into the role of CD45 isoforms in T cell antigen receptor signal transduction was carried out by transfecting CD45-negative CD4 ؉ CD8 ؉ HPB-ALL T cells with the CD45R0, CD45RBC, and CD45RABC isoforms. Fluorescence resonance energy transfer analysis showed that the CD45R0 isoform, but not the CD45RBC or CD45RABC isoforms, was found as homodimers and also preferentially associated with CD4 and CD8 at the cell-surface. A comparison was therefore made of T cell antigen receptor signaling between sub-clones expressing either CD45R0 or CD45RBC. Under basal conditions CD4-associated p56 lck tyrosine kinase activity and cellular protein tyrosine phosphorylation levels were higher in the CD45R0 ؉ than in the CD45RBC ؉ sub-clones. Upon CD3-CD4 ligation, TCR-phosphorylation, ZAP-70 recruitment to the p21/p23 TCR-phosphoisomers, ZAP-70 phosphorylation, as well as p56 lck , c-Cbl and Slp-76 phosphorylation, were all markedly increased in CD45R0 ؉ compared with CD45RBC ؉ cells. T cell antigen receptor (TCR) stimulation alone also promoted c-Cbl phosphorylation in CD45R0 ؉ but not in CD45RBC ؉ cells. Our results are consistent with a model in which association of CD45R0 with CD4 generates a more active pool of CD4-associated p56 lck kinase molecules. Upon CD3-CD4 co-ligation, the active p56 lck increases the intensity of T cell antigen receptor signal transduction coupling by promoting TCR-chain phosphorylation and ZAP-70 recruitment.
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Papers by Janos Szöllosi